Limited representation of intratumoral immune cells is a major barrier to tumor control distinct from the barrier of immunosuppression. Understanding and overcoming this limitation will enable us to extend the effectiveness of many immunotherapies to a broader cross-section of cancer patients. It has been assumed that all tumor- infiltrating lymphocytes are effectors that differentiate in tumor-draining lymph nodes (LN) and subsequently enter the tumor. However, we showed that naive CD8 T cells (TCD8) enter tumors directly, where they differentiate into functional effectors. Naive TCD8 infiltration depends on L-selectin and CCR7, and on the development of tumor blood vessels that express peripheral node addressing (PNAd) and the chemokine CCL21 and resemble LN high endothelial venules. We have also identified organized tertiary lymphoid tissue (TLO) in juxtaposition to this LN-like vasculature. PNAd+ LN-like vasculature has been reported in human solid tumors in association with TLO-like tissue, and correlated with longer metastasis-free, disease-free, and overall survival in breast cancer patients. These data suggest that induction of LN-like vasculature could bolster anti- tumor immunity by enhancing the infiltration and in situ activation of naive TCD8. Induction of high endothelial venules in LN is controlled by lymphotoxin- receptor signaling. However, we found that CCL21 expression depends on IFN? secreted by TCD8 effectors, while PNAd expression is controlled by lymphotoxin-?3. This suggests that early infiltration of effectors induces LN-like vasculature, which in turn supports a self-sustaining recruitment of nave T cells that differentiate within the tumor. The aims of this application are: (1) to determine how TNF receptor ligands regulate the expression of PNAd on tumor vasculature. We will evaluate which scaffolding proteins and enzymes are responsible for tumor associated PNAd. We will also determine if TNF? and lymphotoxin-?3 act redundantly to induce PNAd expression in subcutaneous tumors, since both are ligands for TNF receptors 1 and 2; (2) to determine how the expression of CCL21 in tumors is regulated. We will determine how IFN? controls the expression of CCL21 by gp38+CD31neg cell and endothelial cells in IP tumors. We will also identify mechanisms controlling CCL21 expression in SC tumors; (3) to evaluate the organization and function of TLO-like structures that form in association with LN-like vasculature in tumors. We will evaluate the role of gp38+CD31neg cells as organizers of TLO in tumors, and the factors that influence their activity. We will also determine whether these TLO serve as a locus for positive or negative regulation of anti- tumor immunity. As an R21 application, our purpose is to conduct experiments that will distinguish among different novel mechanisms that control expression of these molecules, enabling development of a future R01 application to investigate means to manipulate them to enhance anti-tumor immunity and cancer control.